PT1411918E - Methods to mobilize progenitor/stem cells - Google Patents

Abstract

Methods to elevate progenitor and stem cell counts in animal subjects using compounds which bind to the chemokine receptor CXCR4 are disclosed. Preferred embodiments of such compounds are of the formula Z-linker-Z' (1) or pharmaceutically acceptable salt thereof wherein Z is a cyclic polyamine containing 9-32 ring members of which 3-8 are nitrogen atoms, said nitrogen atoms separated from each other by at least 2 carbon atoms, and wherein said heterocycle may optionally contain additional heteroatoms besides nitrogen and/or may be fused to an additional ring system; or Z is of formula (a), wherein A comprises a monocyclic or bicyclic fused ring system containing at least one N and B is H or an organic moiety of 1-20 atoms, Z' may be embodied in a form as defined by Z above, or alternatively may be of the formula -N(R)-(CR₂)_n-X wherein each R is independently H or straight, branched or cyclic alkyl (1-6C), n is 1 or 2, and X is an aromatic ring, including heteroaromatic rings, or is a mercaptan; "linker" represents a bond, alkylene (1-6C) or may comprise aryl, fused aryl, oxygen atoms contained in an alkylene chain, or may contain keto groups or nitrogen or sulphur atoms.

Description

&Quot; METHODS FOR MOBILIZING PROGENITOR / STEM CELLS "

Technical Field The invention is in the field of medicinal chemistry and therapeutics. More particularly, the invention relates to methods for mobilizing progenitor / stem cells in individuals by administering certain polyamines.

TECHNICAL BACKGROUND

Blood cells play a crucial role in maintaining the health and viability of animals, including humans. White blood cells include neutrophils, macrophages, eosinophils and basophils / mast cells, as well as the B and T cells of the immune system. White blood cells are continuously replaced by the hematopoietic system, by the action of colony stimulating factors (CSFs) and various cytokines in stem cells and progenitor cells in hematopoietic tissues. Nucleotide sequences encoding a number of these growth factors were cloned and sequenced. Perhaps the most widely known of these is granulocyte colony stimulating factor (G-CSF), which has been approved for use in neutralizing the negative effects of chemotherapy, by stimulating the production of white blood cells and progenitor cells (stem cell mobilization of peripheral blood). A discussion of the hematopoietic effects of this factor can be found, for example, in U.S. Patent No. 5,588,823.

Several other factors have been reported to increase white blood cells and progenitor cells in both human and animal subjects. These agents include macrophage and granulocyte colony stimulating factor (GM-CSF), Interleukin 1 (IL-1), Interleukin 3 (IL-3), Interleukin 8 (IL-8), PIXY-321 (fusion protein GM-CSF / IL-3), macrophage inflammatory protein, stem cell factor, thrombopoietin-related oncogene and growth, as single agents or in combination (Dale, D., et al., Am. J. of Hepnatol. 1998) 57: 7-15, Rosenfeld, C., et al., Bone Marrow Transplantation (1997) 17: 179-183; Pruijt, J., et al., Cur. (1995) 23: 335-340, Broxmeyer, et al., Blood Cells, Molecules and Immunol.

Diseases (1998) 24: 14-30; Glaspy, J., et al. , Cancer Chemother.

Pharmacol. (1996) 38 (suppl.): S53-S57: Vadhan-Raj, S., et al.

Aran. Intermediate. Med. (1997) 126: 673-81; King, A., et al.

Blood (2001) 97: 1534-1542; Glaspy, J., et al. , Blood (1997) 90: 2939-2951).

Although endogenous growth factors are pharmacologically effective, the well-known drawbacks of employing proteins and peptides as medicaments underlie the need to add to the repertoire of such growth factors with agents which are small molecules. In another aspect, such small molecules are advantageous over proteins and peptides, where bulk production is desired.

Over the past several years, various cyclic polyamine antiviral agents have been described in a series of Patents 2 and U.S. applications. These patents are U.S. Patents No. 5021409; 6001826; 5,583,131; 5698546 and 5817807. PCT Publications WO 00/02870, based on a application filed on July 8, 1998 and WO 01/44229, based on a request filed December 17, 1999, which describes additional compounds. These publications describe the structural features of cyclic polyamine antiviral agents.

The structural features of various non-cyclic amine antiviral agents have also been described in a series of U.S. applications, now published as PCT publications. These publications are, WO 00/56729, based on a request filed on March 24, 2000; WO 02/22600, based on applications lodged on 15 and 20 September 2000; WO 02/22599, based on applications filed on 15 and 22 September 2000, as well as WO 02/34745, published 2 May 2002.

In addition, improved methods for preparing some of the cyclic polyamine compounds are described in U.S. Patent Nos. 5,612,478; 5756728; 5801281 and 5606053 and PCT publication WO 02/26721, based on a request filed September 29, 2000.

It has been previously disclosed and disclosed in PCT publication WO 02/58653, based on a request filed on February 1, 2000, that some of the polyamine antiviral agents described in the above mentioned publications have the effect of increasing the white blood cell count . It has now been found that the polyamine antiviral agents described in the above mentioned publications also have the effect of increasing progenitor cells and / or stem cells. Development and maturation of blood cells is a complex process. Mature blood cells are derived from progenitor cells of hematopoietic precursor cells and stem cells present in specific hematopoietic tissues, including bone marrow. Within these environments, before they enter the circulation, hematopoietic cells proliferate and differentiate. The chemokine CXCR4 receptor and its natural ligand, stromal cell-derived factor 1 (SDF-1), appears to be important in this process (for reviews, see Maekawa, T., et al., Internai Med. : 90-100;

Nagasawa, T., et al. , Int. J. Hematol. (2000) 72: 408-411). This is demonstrated by references that CXCR4 or SDF-1 knockout mice exhibit hematopoietic defects (Ma, Q., et al., Proc. Natl Acad Sci USA (1998) 95: 9448-9453; Tachibana, K., et al.

Nature (1998) 393: 591-594; Zou, Y.R., et al., Nature (1998) 393: 595-599). It is also known that CD34 + progenitor cells express CXCR4 and require SDF-1 produced by bone marrow stromal cells for chemoattraction and grafting (Peled, A., et al., Science (1999) 283: 845-848) in vitro, SDF-1 is chemotactic for CD34 + cells (Aiuti, A., et al., J. Exp. Med. (1997) 185: 111-120, Viardot, A., et al., Ann. (1998) 77: 194-197) and for progenitor / stem cells (Jo, DY., Et al., J. Clin. Invest. (2000) 105: 101-111). SDF-1 is also an important chemoattractant, signaling through the CXCR4 receptor, for several other more compromised progenitor and mature blood cells, including T lymphocytes and monocytes (Bleul, C., et al., J. Exp. Med. 1996) 184: 1101-1109), pro- and pre-B lymphocytes (Fedyk, ER, et al., J. Leukoc. Biol. (1999) 66: 667-673; Ma, Q., et al., Immunity (1999) 10: 463-471) and megakaryocytes (Hodohara, K., et al.

Blood (1999) 96: 4142-4151;

Blood (2000) 95: 769-775; Riviere, C., et al., 95: 1511-1523; Majka, M., et al., Blood (2000) 4

Gear, A., et al., Blood (2001) 97: 937-945; Abi-Younes, S., et al. , Circ. iies. (2000) 86: 131-138).

Thus, in brief, it appears that SDF-1 is capable of controlling the positioning and differentiation of cells containing CXCR4 receptors, whether these cells are stem cells (ie, cells that are CD34 +) and / or progenitor cells (resulting in formation of specific types of colonies, in response to particular stimuli, which may be CD34 + or CD34-) or cells that are somewhat more differentiated.

Recently, considerable attention was focused on the number of CD34 + cells mobilized in the peripheral blood progenitor cell cluster, used for autologous stem cell transplantation. The CD34 + population is the component thought to be primarily responsible for improved recovery time following chemotherapy and the cells most likely responsible for long-term graft and reestablishment of hematopoiesis (Croop, JM, et al., Bone Marrow Transplantation 2000) 26: 1271-1279). The mechanism by which CD34 + cells re-express may be due to the chemotactic effects of SDF-1 on cells expressing CXCR4 (Voermans, C. Blood, 2001, 97, 799-804; Ponomaryov, T., et al., J. Clin Invest (2000) 106: 1331-1339). More recently, adult hematopoietic stem cells have been shown to be able to restore damaged heart tissue in mice (Jackson, K., et al., J. Clin Invest. (2001) 107: 1395-1402; Kocher, A., et al.

Nature Med. (2001) 7: 430-436).

Thus, the role of the CXCR4 receptor has assumed considerable significance in cell positioning and differentiation management. 5 The citation of the above documents should not be seen as an admission that any of the above is relevant prior art. All statements regarding the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

DISCLOSURE OF THE INVENTION The invention is directed to methods of obtaining progenitor and / or stem cells from animal individuals, in particular, veterinary and human subjects. Progenitor and / or stem cells can be harvested and used in cell transplantation. The methods of the invention employ the compound 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane or the pharmaceutically acceptable salts and metal complexes thereof.

Accordingly, in one aspect, the invention is directed to a method for obtaining progenitor and / or stem cells from a subject, which method comprises administering to said subject an amount of the above compound or a pharmaceutically acceptable salt or metal complex thereof, sufficient for mobilizing levels of progenitor cells and / or stem cells in the peripheral blood of said subject, followed by collection of said progenitor and / or stem cells. In one embodiment, the bone marrow progenitor and / or stem cells are mobilized for myocardial repair. The invention is also directed to a compound for use in a method for treating a hematopoietic deficit in a subject, wherein said method comprises: (a) administering to said subject an amount of a compound sufficient to mobilize progenitor and / or stem cells in the subject (b) collecting said progenitor and / or stem cells and (c) transplantation of said progenitor and / or stem cells to said subject, said compound being 1,1 '- [1 , 4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane or a pharmaceutically acceptable salt or metal complex thereof.

Preferred forms of the compounds of the invention are discussed below.

Brief Description of the Drawings Figure 1 shows a graph of obtaining myeloid progenitors in response to treatment with 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane (AMD3100) in combination with macrophage inflammatory protein following administration of G-CSF. 7

Modes of Carrying Out the Invention

The compounds useful in the invention are 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane and the pharmaceutically acceptable salts and metal complexes thereof.

The above compounds inhibit HIV replication by inhibiting CXCR4, the co-receptor required for fusion and entry of the T-tropic HIV strains, and also inhibit natural ligand-induced ligand and ligand binding to the SDF-1 chemokine. While binding to any theory is not desired, the above compounds which inhibit the binding of SDF-1 to CXCR4, by virtue of such inhibition, effect an increase in stem and / or progenitor cells. The increase of stem and / or progenitor cells in the blood is useful in treatments to alleviate the effects of protocols that adversely affect the bone marrow, such as those resulting in leucopenia. These are known side effects of chemotherapy and radiation therapy. The above compounds also enhance the success of bone marrow transplantation, improve wound healing and burn treatment, and aid in the restoration of injured organ tissue. They also fight bacterial infections that are prevalent in leukemia. The above compounds are used to mobilize and collect CD34 + cells by means of apheresis, with and without combinations with other mobilizing factors. The harvested cells are used in treatments requiring stem cell transplantation.

As used herein, the term " progenitor cells " refers to cells that, in response to particular stimuli, may form differentiated hematopoietic or myeloid cells. The presence of progenitor cells can be assessed by the ability of the cells in a sample to form colony forming units of various types including, for example, CFU-GM (colony forming units, granulocyte-macrophage); CFU-GEMM (colony forming units, multipotential); BFU-E (explosion-forming units, erythroid); HPP-CFC (highly proliferative potential colonizing cells); or other types of differentiated colonies that can be obtained in culture using known protocols.

As used herein, " stem cells " are less differentiated forms of progenitor cells. Typically, such cells are often CD34-positive. However, some stem cells do not contain this marker. These CD34 + cells can be assayed using fluorescent activation cell sorting (FACS) and, thus, their presence can be evaluated in a sample using this technique.

In general, CD34 + cells are present only at low blood levels, but are present in large numbers in the bone marrow. Although other types of cells, such as endothelial cells and mast cells, may also display this marker, CD34 is considered an index of the presence of stem cells.

Particularly preferred embodiments of the above compounds include 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane, set forth in U.S. Patent No. 5,583,131 and here designated AMD3100.

Methods for synthesizing the compounds useful in the invention are set forth in U.S. patents and applications incorporated herein by reference. 9

As provided above, AMD3100 is an antagonist with the chemokine receptor CXCR4 (Gerlach et al., J. Biol. Chem. (2001) 276: 14153-14160). This compound interferes with the binding of SDF-1 derived from bone marrow stromal cells to CXCR4 in stem cells, which leads to the release into the circulation of hematopoietic stem cells from the bone marrow (Broxmeyer et al., Blood (2001) 98: 811a (Abstract)). In a study of

Phase 1 at the University of Washington, Seattle, a single dose of 80æg / kg of AMD-3100 resulted in a WBC count of 17000 / æl and a maximal increase of 6 fold in circulating CD34 + progenitor / stem cells, in the range of time of 6 hours (Liles et al., Blood 2001 98: 737a (Abstract)). In another recent study, mice were injected with rhG-CSF and recombinant rat Stem Cell Factor (rrSCF) in order to mobilize a large number of bone marrow stem cells into the circulation and then induced a heart attack . The combination of rrSCF and rhG-CSF provides a maximum number of stem cells in circulation after 5 injections daily. At 27 days post surgery, there was a 68% improvement in survival in the treated versus control groups. At this point, dead tissue was replaced with regenerating myocardium and all functional parameters tested were improved as compared to controls (Orlic et al., PNAS (2001) 98: 10344-10349).

The compounds of the invention may be prepared in the form of prodrugs, i.e. protected forms which release the compounds of the invention upon administration to the subject. Typically, the protecting groups are hydrolyzed in body fluids, such as in the blood stream, thereby releasing the active compound or are oxidized or reduced in vivo to release the active compound. A discussion of prodrugs is found in 10

Smith and Williams Introduction to the Principles of Drug Design, Smith, H.J .; Wright, 2nd ed., London (1988).

The compounds of the invention, such as polyamines, may be administered in the form of their acid addition salts or metal complexes. Suitable acid addition salts include salts of inorganic acids which are biocompatible, including HCl, HBr, sulfuric, phosphoric and the like, as well as organic acids such as acetic, propionic, butyric and the like, as well as acids containing more than one carboxyl group such as oxalic, glutaric, adipic and the like. Typically, at physiological pH, the compounds of the invention will be in the forms of the acid addition salts. Hydrochlorides are particularly preferred. In addition, when prepared as purified forms, the compounds may also be crystallized as the hydrates.

The compounds of the invention may be administered as single active ingredients and / or in admixture with additional active ingredients which are therapeutically or nutritionally useful, such as antibiotics, vitamins, aromatic plant extracts, anti-inflammatories, glucose, antipyretics, analgesics, stimulation factor (IL-1), Interleukin-3 (IL-3), Interleukin-8 (IL-8), PIXY-321 (GM-CSF / IL-3 fusion protein) ), macrophage inflammatory protein, stem cell factor, thrombopoietin, growth-related oncogene or chemotherapy, and the like.

The compounds of the invention may be formulated for administration to an animal subject, using generally understood formulation techniques well known in the art. 11

Formulations which are suitable for particular modes of administration and for the above compounds can be found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA.

Preferably, the compounds are administered by injection, most preferably by intravenous injection, but also by subcutaneous or intraperitoneal injection and the like. Additional parenteral routes of administration include intramuscular and intraarticular injection. For intravenous or parenteral administration, the compounds are formulated in the appropriate liquid form with excipients, as required. The compositions may contain liposomes or other suitable carriers. For intravenous injection, the solution is made isotonic using standard preparations, such as Hank's solution.

In addition to the injection, other routes of administration may also be used. The compounds may be formulated into tablets, capsules, syrups, powders or other forms suitable for oral administration. Through the use of suitable excipients, these compounds may also be administered through the mucosa using suppositories or intranasal sprays. Transdermal administration may also be effected by the use of suitable penetrants and the rate of release. The formulation and route of administration chosen will be adapted to the individual in question, the nature of the condition being treated in the individual and, in general, the opinion of the attending physician. 12

Suitable dosage ranges for the above compounds vary according to these considerations, but in general the compounds are administered in the range of about 0.1 pg / kg-5 mg / kg body weight; the range is preferably about 1 pg / kg-300 pg / kg body weight; more preferably about 10æg / kg-100æg / kg body weight. Thus, for a typical 70 kg human subject, the dosage range is about 0.7 pg-350 mg; preferably about 700 pg-21 mg; most preferably about 700 pg-7 mg. The dosages may be higher when the compounds are administered orally or transdermally, as compared to, for example, i.v.

The compounds may be administered as a single dose of bolus, a dose over time, as in i.v. or transdermal or multidose administration.

In addition to direct administration to the subject, the above compounds may be used in ex vivo treatment protocols to prepare cell cultures which are then used to replenish the individual's blood cells. Ex vivo treatment may be conducted in autologous cells collected from peripheral blood or bone marrow or allografts from compatible donors. The concentration of the above compound or compounds, alone or in combination with other agents, such as the macrophage inflammatory protein, is a matter of routine optimization.

Individuals who will respond favorably to the method of the invention include medical and veterinary subjects including, in general, human patients. Among others, those for whom the methods of the invention are useful are cats, dogs, large animals, birds, such as chickens and the like. In general, any subject benefiting from elevation of progenitor cells and / or stem cells, or whose progenitor cells and / or stem cells are desirable for stem cell transplantation, is suitable for administration of the method of the invention.

Typical conditions that may be ameliorated or otherwise benefited by the invention include hematopoietic disorders such as aplastic anemia, leukemias, drug-induced anaemias and hematopoietic deficits of chemotherapy or radiation therapy. The invention is also useful in enhancing the success of transplantation during and after immunosuppressive treatments, as well as in more efficient wound healing and bacterial inflammation treatment. The present invention is still useful for the treatment of individuals who are immunocompromised or whose immune system is otherwise weakened. Typical conditions that are improved or otherwise benefited by the present invention include those individuals who are infected with a retrovirus and, more specifically, who are infected with the human immunodeficiency virus (HIV). Accordingly, the invention is directed to a broad spectrum of conditions for which elevation of progenitor cells and / or stem cells in a subject would be beneficial or where collection of progenitor cells and / or stem cells for subsequent stem cell transplantation would be beneficial.

The compounds of the invention are also administered to regenerate the myocardium through the mobilization of bone marrow stem cells. 14

Since the invention has now been generally described, it will be more readily understood by reference to the following examples which are provided by way of illustration and unless otherwise specified are not intended to be limiting of the present invention.

Example 1

Elevation of Mouse Progenitor Cells

The effects of subcutaneous (sc) administration of 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane (AMD3100) on C3H / H3 J mice (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitors per ml of blood. The progenitors were stimulated to form colonies in vitro, with the combination of 1 U / ml Epo rhu, 50 ng / ml SLF rhu, 5%, Vol / Vol, of mouse spleen mitogenic cell conditioned medium dyer herb (PWMSCM) and 0.1 mM hemin. Plates were scored 7 days after incubation.

The time-dependent effects on the number of progenitors mobilized with AMD3100 are for a single 5 mg / kg s.c. injection and are shown in Table 1.

Table 1

Absolute Progenitors By ML of Blood Methyl Cellulose Culture

CFU-GM BFU-E CFU-GEMM

Control 289.8 49.4 25.8 AMD3100: 15 " 791.6 134.5 90.4 AMD3100: 30 " 1805.5 209.3 113.5 AMD3100: 120 " 828.7 102.3 47.6

To measure dose-dependent effects, AMD3100 was administered at 1, 2.5, 5 and 10 mg / kg by a single sc injection and the number of progenitors per ml of blood was measured 1 hour after administration and the results are shown in Table 2.

Table 2 Absolute Culture Number Progenitors Per ML of Methylcellulose Blood CFU-GM BFU-E CFU-GEMM Saline Solution 188, 1 16 19 AMD3100: 10 mg / kg 825, 6 120.5 79.8 AMD3100: 5 mg / kg 608, 4 92.8 69.5 AMD3100: 2.5 mg / kg 687, 6 98.9 70.6 AMD3100: 1 mg / kg 424 62 27.1 16

Change Factor Compared to Time 0

Progenitors Culture of Methylcellulose Time GM BFU-E CFU-GEMM 15 " 2.73 2.72 3.51 30 " 6.23 4.24 4.41 2 '2.86 2.07 1.85 Maximum mobilization of mouse progenitors is achieved at a dose of 2.5 to 10 mg / kg of AMD3100, approximately 0.5 to 1 hour after injection, as shown in Table 3.

Example 2

Mobilization of Mouse Progenitor Cells in Combination with ΜΙΡ-lot and G-CSF The mobilization capacity of AMD3100 progenitor cells in combination with mouse (mu) macrophages inflammatory protein (MIP-1α) was tested with or without administration of G-CSF rhu. It has been previously shown that ΜΙΡ-Ια mobilizes progenitor cells in mice and humans (Broxmeyer, E., et al., Blood Cells, Molecules, and Diseases (1998) 24 (2): 14-30).

Groups of mice were randomized to receive either control diluent (saline solution) or G-CSF at a dose of 2.5 pg per mouse twice daily for two days by injecting the sc 11 hours after final injection of saline or G-CSF, the mice were divided into clusters to receive IV MIP-1I administered at a total dose of 5æg, AMD3100, administered sc, at a dose of 5 mg / kg or a combination of MIP-1α and AMD3100 at the same doses. One hour later, the mice were sacrificed and the number of progenitor cells per ml of blood was measured. These data are summarized in Figure 1. The AMD3100 acts in an additive mode over the additive for mobilization of progenitor cells, when used in combination with mouse macrophages (MIP) -I1I inflammatory protein, each administered 11 hours after addition of rhu of G-CSF or control diluent (saline) and 1 hour prior to blood evaluation.

Example 3

Clinical Elevation of Progenitor Cells

Five healthy human volunteers were used in the study, with baseline white blood cell counts of 4500-7500 cells / mm3. Each patient received a single subcutaneous (sc) injection of 80 pg / kg of AMD3100 (ie, 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11 -tetraazacyclotetradecane) in 0.9% saline solution from a stock solution of 10 mg / mL AMD3100 in brine under sterile conditions. Blood samples were obtained by catheter, before the dose and at various times up to 24 hours after dosing. 18

Blood samples were evaluated in terms of total white blood cells, CD34 positive progenitor cells (by FACS analysis) as a percentage of total white blood cells, as well as the absolute number per mL and cycle status of macrophage progenitor cells and granulocytes (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM).

As shown in Tables 3 and 4, administration of AMD3100 caused elevation of white cell count and CD34-positive progenitor cells in human volunteers, which maximized at 6 hours post-dose.

Table 3

AMD3100-induced mobilization of white blood cells in volunteers (x 103 WBC). ID Trace Baseline TREATMENT 30 Min 1 H 2 H 4 H 6 H 9 H Day 2 PI 7.4 6.41 8.02 14.8 21.4 23.2 26.2 22.3 7.07 P2 6 , 04 5, 45 6.53 8.93 13.5 18.00 19.2 19.6 8.03 P3 4.38 5, 8 7, 14 9.28 ND 18, 10 17, , 98 P4 5.08 5.31 4.37 7.38 12.4 14.6 15.8 13.9 4.98 P5 4. 53 5.02 6.08 8.43 ND 16.90 19.3 19.00 4, 57 19

Table 4

AMD3100-induced mobilization of CD34-positive cells, expressed as the percentage of total WBCs in volunteers. ID Baseline TREATMENT 1 H 3 H 6 H 9 H Day 2 PI 0.07 0.04 0.07 0.11 0.18 0.08 P2 0.08 0.06 0.08 0.13 0.11 0.12 P3 0.07 0.16 0.06 ND 0.11 0.07 P4 0.05 0.07 0.09 0.09 0.1 0.1 P5 0.12 0.12 0.13 0 , 2 0.2 0.16 The blood was also analyzed in terms of the AMD3100 having mobilized these parents.

The absolute number of unseparated and low density nucleated cells (separated by Fico-hypaque), per ml of blood, as well as the absolute number per ml and cycle status of macrophages and granulocyte progenitor cells (CFU-GM) , erythroid (BFU-E) and multipotential (CFU-GEMM) in normal donors injected sc with AMD3100. The above parameters were evaluated prior to injection and at 1, 3, 6, 9 and 24 hours after AMD3100 injection. All progenitor cell results are based on the score of 3 culture plates per assay, per point.

As for the number and cycle status of progenitor cells, the number of CFU-GM, BFU-E and CFU-GEMM in methylcellulose cultures, by stimulation of the cells with 1 unit 20 (U) / ml recombinant human erythropoietin (rhu) , 100 U / ml of macrophages and granulocyte colony stimulating factor rhu (GM-CSF), 100 U / ml interleukin-3 rhu (IL-3) and 50 ng / ml Steel factor rhu = stem cell factor (SCF)). CFU-GM were also evaluated in agar cultures stimulated with 100 U / ml rhu GM-CSF and 50 ng / mL rhu of SLF. For both types of assays, colonies were scored after incubation for 14 days in a humidified atmosphere with 5% CO2 and decreased O2 tension (5%). The parental cell cycle status was measured using a high activity specific tritiated thymidine killing technique as previously described (Broxmeyer, E., et al., Exp. Hematol. (1989) 17: 455-459) .

The results are given first as the mean change factor in the absolute number of nucleated cells and progenitors at 1, 3, 6, 9 and 24 hours, as compared to pre-injection counts (= Time (T) 0) for all five donors, as seen in Tables 5-7.

In the tables below, STD - standard deviation STE - standard error PBL-US - non-separated peripheral blood PBL-LD - low density peripheral blood (Separated with

Ficoll) P - Significance using a bilateral t-test 21

Table 5

(Mean of 5 donors) PBL- -US PBL- -LD AVERAGE STD STE% CHG P AVERAGE STD STE% CHG P o II H 1.00 0.00 0.00 0, 0% 1.00 0.00 0.00 0.0% T = 1 1, 69 0.00 0.00 68.6% 0.017 1.86 0.00 0.00 86.2% 0,000 cn II H 2, 80 0.51 0.23 180.2% 0.000 2.86 0.28 0.12 185.6% 0.000 II H 3, 26 0.61 0.27 225.8% 0.000 3.66 0, 43 0.19 266.3% 0.001 II H 3, 09 0.89 0.31 209.4% 0.000 3.64 1.18 0.53 264.3% 0.001 T = 2 4 1 , 07 0.85 0.29 7.0% 0.553 1.05 1, 19 0.53 4.6% 0.815 22

Table 6 CULTURE OF METILCELULOSE CFU-GM BFU-E CFU-GEMM AVERAGE STD STE% CHG P AVERAGE STD STE% CHG P AVERAGE STD STE% CHG P τ = ο 1.00 0.00 0.00 0.0¾ 1.00 0.00 0.00 0.0¾ 1.00 0.00 0.00 0.0¾ Τ = 1 4.77 0.00 0.00 376.7¾ 0.001 1.99 0.00 0.00 98.9¾ 0.002 2.32 0.00 0.00 131.8¾ 0.000 Τ = 3 13.66 1.56 0.70 1266.5¾ 0.001 3.21 0.50 0.22 221.3¾ 0.004 433 0.44 0.20 332 , 5¾ 0.000 Τ = 6 21.71 5.78 2.58 2070.6¾ 0.000 8.01 1.25 0.56 500.5¾ 0.008 10.07 0.59 0.27 907.2¾ 0.002 Τ = 9 10, 47 5.09 228 947.3¾ 0.000 4.34 2.99 1.34 334.4¾ 0.000 5.25 4.54 2.03 425.4¾ 0.014 Τ = 24 1.58 3.01 1.34 65.5¾ 0.005 1.26 1.02 0.45 26.3¾ 0.194 1.53 3.04 1.36 53.2¾ 0.199

Table 7 CFU-GM IN AGAR CULTURE STD STE% CHG P o II H 1.00 0.00 0.00 0.00% T = 1 2.81 0.00 0.00 180.8% 0.001 T = 3 8.54 0.75 0.34 754.1% 0.000 T = 6 17.93 1.62 0.72 1692.8% 0.000 II H 10.25 4.57 2.04 924.9% 0.000 T = 2 4 2.08 2.06 1.03 108.3% 0.073

The results are then shown as a factor of changing the levels of T = 0 for each individual donor, as shown in Tables 8-10.

Table 8 COMPARATIVE CHANGE FACTOR WITH TIME = 0 for each individual patient (P) NUCLEAR CELLULARITY PBL-US PBL-LD PI P2 P3 P4 P5 PI P2 P3 P4 P5 or II H 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 H II 2.54 138 1.38 1.36 1.78 2, 07 1.99 1, 48 1.66 2.10 T = 3 3.55 2.74 2, 02 2, 48 3.23 2.83 3.25 2, 17 2.82 3.20 T = 6 3.97 2.94 2.74 2.60 4. 044 , 07 3.90 227 2.78 5.30 II H 3.27 330 2.69 2.24 3.96 3.65 4.43 2.47 2.48 5.17 T = 2 4 1.21 1 , 43 0.96 0, 77 0.99 1, 01 1, 71 0.79 0.60 1, 12 2 4

Table 9

PROGENITORS

Table 10 CFU-GM IN AGAR CULTURE Pl P2 P3 P4 P5 or II H 1.00 1.00 1.00 1.00 1.00 T = 1 3.05 3.74 1.67 2.71 2.87 T = 3 8.88 9.49 7, 47 10.46 6.40 T = 6 17, 77 24, 01 14, 04 13.07 20.75 II H 1028 7, 72 10.22 12.78 T = 2 4 3.69 1, 13 1.30 2.20 The actual number of nucleated cells and progenitor cells per ml of blood and the cycling state (=% of progenitors in the (S) phase of cell cycle DNA synthesis) of progenitors for each of the five donors (No. Pl, P2, P3, P4 and P5) is shown in Tables 11 and 12.

Table 11 CFD-GM BFD-E PI CFU-GEMM CFD-GM BFD-E P2 CFD-GEMM Ν ° Absolute of Progenitors per 1.1 L Cycle Status of Progenitors Absolute No. of Progenitors per 1.1 L Cycle Status of Progenitors Absolute No. of Progenitors per 1.1 L Progenitor Cycle Status Absolute No. of Progenitors per 1.1 L Progenitor Cycle Status Absolute No. of Progenitors per 1.1 L Progenitor Cycle Status Absolute No. of Progenitors per 1.1 L Cycle of Progenitors τ = ο 241 6¾ 261 0¾ 121 68 213 08 410 28 120 08 Τ = 1 1259 1¾ 614 0¾ 264 08 1455 08 608 38 272 68 Τ = 3 1160 1¾ 1340 138 540 18 4646 28 809 08 418 08 Τ = 6 3624 0¾ 2388 08 949 08 6540 08 1502 08 1126 08 1 = 9 1541 2¾ 1418 118 335 08 3416 08 1886 08 854 48 Τ = 2 4 211 0¾ 218 08 142 08 521 38 168 28 316 08 CFD-GM BFD-E CFD-GEMM CFD-GM BFD-E CFD-GEMM P3 P4 Absolute State Number of State Absolute Absolute State Absolute Absolute State N ° Absolute of State of N Absolute Progenitor Status Progenitor Cycle Progenitor Cycle Progenitor Cycle by Cycle of Progenitors per Cycle of Progenitors Cycle by 1.1 L Progenitors by 1.1 L Progenitors by 1.1 L Progenitors 1.1 L Progenitors 1.1 L Progenitors per 1.1 L Progenitors T = 0 281 08 351 08 140 08 138 08 460 08 101 08 T = 1 1040 08 808 08 312 08 941 08 612 08 199 08 1 = 3 4233 18 915 08 610 08 2851 58 1195 08 519 08 1 = 6 5895 08 1593 08 916 08 3936 08 1533 08 920 88 T = 9 2641 08 1302 08 341 08 1942 08 1348 08 451 08 T = 24 402 08 402 08 91 08 208 58 362 38 99 08 CFD- GM BFD- E CFD-GEMM P5 Absolute State Number Absolute State Absolute State Number of Progenitors Progenitors Cycle Progenitors Cycle per 1.1 L Progenitors per 1.1 L Progenitors per 1, 1 L Progenitors 1 = 0 169 08 343 18 55 08 1 = 1 481 08 130 08 169 08 T = 3 1423 58 1288 38 244 08 T = 6 3454 08 3208 18 981 08 T = 9 1110 08 1131 08 526 08 T = 24 310 08 495 08 12 4 08

Table 12 CFU-GM in AGAR Culture CFU-GM in AGAR Culture CFU-GM in AGAR Culture CFU-GM in AGAR Culture CFU-GM in AGAR Culture Pl P2 P3 P4 P5 Absolute N ° Absolute N ° State State of Absolute N ° State of Absolute N ° State of Absolute State of Cycle of Cycle of Cycle of Cycle of Cycle of Progenitors Progenitors Progenitors Progenitors Progenitors Progenitors Progenitors Progenitors Progenitors Progenitors Progenitors Progenitors by 1.1 L by 1, 1 L per 1.1 L per 1.1 L per 1.1 L τ = ο 233 6¾ 100 0¾ 140 0¾ 124 0¾ 104 0¾ Τ = 1 710 0¾ 376 0¾ 234 0¾ 336 0¾ 298 3¾ Τ = 3 2070 0¾ 953 1¾ 1049 0¾ 1299 0¾ 664 0¾ Τ = 6 4142 0¾ 2409 3¾ 1972 3¾ 1623 0¾ 2153 1¾ Τ = 9 1032 0¾ 1085 0¾ 1268 0¾ 1326 0¾ Τ = 24 371 0¾ 159 0¾ 162 0¾ 229 0¾

Results for all five donors were very consistent with the maximum factors of increases in circulating levels of progenitor cells found 6 hours after injection of AMD3100 in human donor subjects. The progenitors were in a slow-cycle or non-loop state before and 1, 3, 6, 9 and 24 hours after AMD3100 injection.

Example 4 Membrane Stem Cells Mobilized for Myocardial Repair

Adult rats are anesthetized and a thoracotomy is performed. The descending branch of the left coronary artery is lacquered and not reperfused. Within 4 to 6 hours after lacquering, animals are injected with limit dilution of AMD-3100 or AMD-3100 plus rhG-CSF. Control mice are not treated with the reagents. The animals are monitored, at intervals of one week, by echocardiography and MRI. The experiment is finished at 2, 6 to 12 weeks after surgery. On the day of sacrifice, hemodynamic functions are analyzed in terms of left ventricular end-diastolic pressure, developed left ventricular pressure, and rate of rise and fall of left ventricular pressure. The heart is then stopped in diastole and perfused through the abdominal aorta to clear residual blood from the myocardial vascular network. This is followed by perfusion of the heart with 10% formalin. Several cuts are made through the fixed heart and these are embedded in paraffin and sections. Sections are stained and analyzed by light microscopy to determine infarct size in treated and control animals. The 29 tissue sections of hearts withdrawn at 2 weeks post surgery are stained with specific antibodies to immature developing myocytes and blood vessel proteins and analyzed by confocal microscopy. Immunohistochemical analysis involves the identification of transcription factors and surface markers, expressed in early stages of myocyte development. The results of this experiment will show that when the AMD-3100 reagent is administered within hours of induction of cardiac ischemia, together with or without rhG-CSF, this reagent rapidly mobilizes bone marrow stem cells and results in a blockage to cardiac remodeling and formation and will lead to the regeneration of the dead myocardium.

Lisbon, February 24, 2012 30

Claims (18)

A method of obtaining progenitor and / or stem cells from a subject, which method comprises (a) administering to said subject an amount of a compound sufficient to mobilize said progenitor and / or stem cells in the peripheral blood of said subject, followed (b) collecting said progenitor and / or stem cells, wherein said compound is 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11- tetraazacyclotetradecane or a pharmaceutically acceptable salt or metal complex thereof. The method of claim 1, wherein the compound is 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane. The method of claim 3, wherein the compound is a hydrochloride salt of 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane. 1 The method of claim 1, wherein the compound is a metal complex of 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane. The method of any preceding claim, wherein the progenitor and / or stem cells are harvested by apheresis. The method of any preceding claim further comprising administering inflammatory macrophage protein in the peripheral blood or bone marrow of said subject. The method of any preceding claim further comprising administration of G-CSF in the peripheral blood or bone marrow of said subject. A compound for use in a method of treating a hematopoietic deficit in a subject, wherein said method comprises (a) administering to said subject an amount of a compound sufficient to mobilize progenitor and / or stem cells in the peripheral blood of said subject, b) collecting said progenitor and / or stem cells and (c) transplantation of said progenitor and / or stem cells to said subject, said compound being 1,1 '- [1,4-phenylene-bis - (methylene)] bis-1,4,8,11-tetraazacyclotetradecane or a pharmaceutically acceptable salt or metal complex thereof. The compound for use of claim 9, wherein the compound is 1,1 [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane. The compound for use of claim 9, wherein the compound is a pharmaceutically acceptable salt of 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane. The compound for use of claim 11, wherein the compound is a 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane hydrochloride salt. The compound for use of claim 9, wherein the compound is a metal complex of 1,1 '- [1,4-phenylene-bis (methylene)] bis-1,4,8,11-tetraazacyclotetradecane. The compound for use of any one of claims 9-13, wherein said hematopoietic deficit is a result of chemotherapy. The compound for use of any one of claims 9-13, wherein said hematopoietic deficit is a result of radiation therapy. The compound for use of any one of claims 9-15, wherein the progenitor and / or stem cells are harvested by apheresis. 3 The compound for use of any one of claims 9-16, wherein said method further comprises administering macrophage inflammatory protein in the peripheral blood or bone marrow of said subject. The compound for use of any one of claims 9-17, wherein said method further comprises administering G-CSF in the peripheral blood or bone marrow of said subject. Lisbon, February 24, 2012 4